This is a Career Development Award-2 (CDA-2) application by Jae Dugan, Ph.D., mentored by Michael Davey, M.D., Ph.D. and Daniel Carr, Ph.D. The PI developed expertise in molecular bacteriology during his thesis work investigating mechanisms of antibiotic resistance in Chlamydia. His current research interests involve understanding how the innate immune system contributes to human disease. Toll-like receptors (TLR) and NOD-like receptors (NLR) function as "sensing" proteins to activate the innate immune system. This application focuses on one member of the NLR family called nucleotide oligomerization domain 2 (NOD2). Mutations in NOD2 are linked to Crohn's disease, sarcoidosis, asthma, atopy, peritonitis, graft- versus-host disease, leprosy and Blau syndrome (chronic granulomatous inflammation of the skin, eyes, and joints). One specific domain within the NOD2 protein is the nucleotide oligomerization domain (NOD), also referred to as the nucleotide binding domain (NBD). Mutations found in Blau syndrome cluster in the NOD domain (the most frequent being a glutamine at position 334 instead of an arginine, indicated as R334Q). Studies of the NOD domain in other NLR family members have shown this domain to have an ATP binding site, leading to hydrolysis of ATP, followed by self oligomerization. Oligomerization is thought to be critical for subsequent downstream events. Blau syndrome represents a unique opportunity to investigate how NOD2 can contribute to inflammatory disease in multiple organs. The work proposed here will test the following hypotheses: 1. The R334Q-NOD2 mutation causes increased ATP binding, hydrolysis and oligomerization of NOD2 compared to wild type protein 2. Macrophages from mice expressing one copy of the equivalent of the human mutation will have increased cytokine release, intracelular signalling and autophagy formation compared to wild type mice and 3. Mutant mice will show signs of spontaneous inflammation, or enhanced inflammatory responses after exposure to known activators of NOD2. These hypotheses will be tested in 3 aims. In aim 1 the PI will investigate the impact of the R334Q mutation by studying human NOD2 expressed in and purified from insect cells. ATP binding and hydrolysis and oligomerization of NOD2 will be tested using wild type and R334Q-NOD2. Aims 2 and 3 will employ a knock-in mouse created by the PI and described in the work accomplished section where one copy of NOD2 carries the murine equivalent (R314Q) of the human R334Q mutation. Macrophages prepared from bone marrow and peritoneal cavity will be studied for cytokine response, intracellular signalling and autophagosome formation in response to muramyl dipeptide (MDP) a known activator of NOD2 (aim 2). Studies of synergy will also be performed with MDP and TLR agonists. Mutant mice will be carefully studied for signs of spontaneous inflamamtion and cytokine responses will be followed in sera following iv injection of MDP, MDP and TLR agonists, TLR agonists, and liver bacteria (aim 3). These studies have the potential to provide new and important insights into the mechanism by which NOD2 contributes to chronic inflammation. These insights should lead to rational, targeted interventions. The aims of this application also provide for a broad training experience for Dr. Dugan, greatly expanding his set of skills as he transitions to independence. PUBLIC HEALTH RELEVANCE: NOD2 has been implicated in many chronic inflammatory conditions including Crohn's Disease, sarcoidosis, atopy, peritonitis and graft-versus-host disease. These diseases are important in veteran's health. Our understanding of diseases mediated by innate immunity is just in the early stages of development. Understanding how NOD2 controls the development of inflammation leading to tissue injury and pathology will have broad implications for the health of veterans.